This is a revised R21 grant application submitted in response to Program Announcement PA-03-107 which solicits applications for exploratory/developmental projects. The goal of this project is to develop a transient genetic transformation system for Cryptosporidiumparvum. Genetic transformation is an essential tool for analyzing gene function, but is not available for C. parvum. Together with the almost completed sequence of the C. parvum genome, genetic transformation is needed to translate genomic sequence into biologically and clinically relevant information by elucidating the function of unknown genes and identifying regulatory sequences. We propose to identify genes which are highly expressed in trophozoites and early meronts and insert upstream and downstream intergenic regions from these genes into plasmids carrying the green fluorescent protein reporter. Electroporation methods for the transient transformation of C. parvum sporozoites will be developed with these plasmids. Transformed C. parvum will be identified directly by fluorescent microscopical analysis of infected cell cultures. Alternatively, GFP specific antibodies or reversetranscription PCR will be used. Consistent with the goals of this Program Announcement, this project is exploratory/developmental and is not hypothesis-driven. The goal is the development of a new technique relevant to the study of a pathogenic microorganism. The specific aims are: 1. Identify C parvum genes upregulated in trophozoites/early meronts and construct transient transformation vectors incorporating regions regulating the expression of these genes. Approach: rnRNA transcripts expressed at high level in trophozoites and early meronts will be identified by quantitative real-time PCR. Intergenic regions flanking these genes will be inserted into GFP expression plasmids. 2. Establish a transient transformation system for Cryptosporidium parvum. Approach: Transformation by electroporation of sporozoites with GFP expression plasmids; detection of transformants in cell culture by fluorescent microscopy, anti-GFP antibodies, or reverse-transcriptase PCR.